Could a blood test predict how cancer spreads in children?

An interview with Dr. David Lyden, Stavros S. Niarchos Professor in Pediatric Cardiology and a Professor of Pediatrics at Weill Cornell Medicine, conducted by April Cashin-Garbutt, MA (Cantab).

What methods are currently used to try to predict how cancer will spread in children and adults?

Traditionally, we use biopsies to try to predict how cancer spreads. There are a number of molecular markers that are used, but biopsy is a limited approach. Now, the current trend is to use liquid biopsy, which is basically a blood sample.

Over the last several years, there's been a focus on circulating tumor cells. That whole field is complicated by the fact that when you take a blood specimen such as a tube of blood, it may contain only five to seven circulating tumor cells. It's therefore hard to tell whether cells are really tumor circulating cells; some people think they might be tumor stem cells.

The characterization of these cells is being debated right now in terms of which markers can truly identify these cells and the fact that the cell number is still limited, irrespective of the marker used.

The second popular method that people are using right now is circulating DNA because they want to identify the particular tumor mutation. However, one problem with that is that we have a very high normal cell death rate in our bodies each day and there's DNA being brought into the circulation, even in people without cancer.

When you analyze circulating DNA, 95% of it is coming from normal cell death, which complicates the ability to establish what is tumor DNA, which is the minority, and which is normal DNA that's just coming from normal dying cells.

Our approach is a little bit different. We believe we have a better chance of representing the tumor and the metastatic status of the patient by examining tumor exosomes, which are nanoparticle, barrel-like structures.

The tumor releases billions of these exosomes into the circulation. We are faced with a challenge in that normal exosomes are also being released, in someone without cancer, mainly from the bone marrow cells. However, what we have found over the last 10 years in my laboratory, and what other people's groups have found, is that tumor exosomes are very distinct from normal exosomes.

For instance, normal exosomes have very little to no DNA packaged inside them, whereas tumor exosomes do have DNA packaged inside. Our laboratory was instrumental in identifying double stranded DNA in the exosome.

We are also focused in the laboratory on examining the proteins and lipids and other labs are focusing on the microRNA. We're interested in identifying all the components that compose an exosome. An exosome is exactly like a virus; it has a double lipid bi-layer, it contains trans-membrane proteins that stick out of the exosome and it also contains packaged tumor DNA and RNA inside.

The proteins expressed on tumor exosomes can be in their thousands and they're very different from the few proteins that are packaged in normal exosomes. We want to improve our technology in isolating tumor exosome specifically, but, even if we don't, when you have a mix, it's clear that the tumor exosomes have distinct proteins and tumor DNA present inside them.

The Sohn Conference Foundation recently made a grant to researchers from Weill Cornell Medicine and Memorial Sloan Kettering Cancer Center (MSK) to develop a blood test to predict how cancer spreads in children. What impact would such a test have?

There's not a lot of emphasis on pediatric cancer and metastasis. Most of the focus is on adult cancers and metastasis. Even though pediatric cancers represent 10% of all cancers, it benefits from less than 1% of all NIH and other foundation funding.

I think the Sohn Conference Foundation has focused on this discrepancy and recognized that more funding is needed for children with cancer and metastasis.

What is great about this blood test, which is a liquid biopsy, is that we can easily isolate the exosomes, which are located in the plasma. We perform several ultracentrifugation steps and once we have the exosomes, we can then analyze them for DNA, proteins, lipids and RNA.

The impact of this test is that once we isolate the tumor exosomes, they could help us right from the stage of diagnosis and throughout treatment. In all cases of cancer, including adults with cancer, we never know which patients are going to progress to metastatic diseases. We don't have good predictors of metastasis.

A large proportion of children with neuroblastoma go on to develop bone metastasis; patients with medulloblastoma, a brain tumor in the cerebellum, go on to develop spinal cord metastasis. And with tumors such as osteosarcoma, which is bone cancer, a large percentage of those patients go on to develop lung metastasis. How do we determine who to treat appropriately? We also want to avoid over-treating those patients. For example, perhaps patients without metastasis could benefit from surgery alone.

We feel with the exosome, that certain proteins such as integrins and certain mutations found in the DNA might be helpful for prognosis and tell us which patient will go on. They could also help in the tailoring of therapy. Even with traditional therapies such as chemotherapy, we can measure tumor exosomes and see if the patient is responding to that treatment. Our overall goal with the Sohn Conference Foundation is to identify new proteins that are highly expressed on tumor exosomes, but not in normal tissue, in order to reduce side effects.

Our goal is to develop new drugs and prevent tumor exosomes from binding to distant cells such as lung cells. The proteins on the exosome help with binding or adhesion, and the lipid component helps with fusion, so the tumor exosomes can get into normal lungs and cells, and create a micro environment that's pro-inflammatory that now is favorable for tumor cell binding. We call it a pre-metastatic niche: an environment that's now favorable for metastatic disease.

Furthermore, we are excited about the possibility of identifying the mutational status of a primary tumor. Our goal is to work with Dr. Michael Berger who runs a laboratory at Memorial Sloan Kettering Cancer Center (MSK) called IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets), where they have a test that can tell you the number of mutations in a primary tumor sample - that's over 400 mutations. We want to know whether we can isolate the exosome DNA in the blood test and also identify the mutation; the test will help us confirm.

Now, there are new drugs that are actually specific in blocking the mutation. For example, there's the BRAF mutation and if we use a BRAF inhibitor we can now test the blood to see whether the exosomal DNA is no longer expressing that BRAF mutation. If that were the case, the patient would be responding to therapy.

If we then identify that there is BRAF mutation again in the exosome, that means the patient is now resistant to the therapy and another therapeutic approach should be used. We used to use traditional radiographic scans. Our goal is to identify those patients not responding to a therapy as early as possible, switch them to an improved therapy, and not wait for full blown metastatic disease and, say, a CT scan of the lung.

We think we can improve therapies by identifying these mutations and hopefully developing new drugs that are specific to pediatric cancers, mutations that are unique to those pediatric cancers that haven't been recognized in adult cancers.

What will be the main challenges in developing the test?

There's a difference between composition of tumor exosomes and normal exosomes. We have to do a very good job of identifying specific tumor proteins that are only found in the tumor exosomes and not in normal ones. We have to find whether there is a way that we can specifically isolate tumor exosomes from a blood sample, as opposed to just the summation of all exosomes.

So, the goal in our laboratory is to improve prognostic tests and treatments. We need to have better ways of purifying tumor exosomes specifically and we're fairly successful in isolating exosomes from the blood right now.

The goal, with the aid of the study, is to improve our isolation technique of tumor exosomes, which we think can be achieved.

How will you identify biomarkers that predict future metastasis?

Normal exosomes don't have DNA, whereas tumor exosomes have DNA. Just the presence of DNA alone means that tumor cells are shedding tumor exosomes, but, importantly, I think our best biomarkers are at the protein level, since most of them are transmembrane proteins. These proteins stick out of the exosome, meaning we can use protein mass spectrometry to identify all those proteins expressed on tumor exosomes that could be useful - proteins specific to the tumor that are found on tumor exosomes but never on normal exosomes.

We can see those specific proteins that would help us with prognosis and treatment response. For instance, tumor integrins proteins or adhesion proteins are present on tumor exosomes, but they have limited expression on normal exosomes. If we saw these integrinss, that would indicate that there's tumor progression.

Also the family of integrins is made up of 24 members and certain integrins like to bind to different tissue sites such as the lung, liver, brain and bone. We can actually use those integrins to predict what the future sites of metastasis will be.

What impact will the support of the Sohn Conference Foundation have on your research?

There are very few, if any, grants dedicated to pediatric cancer. Their support alone will therefore have a huge impact. I think the greatest impact is that we'll be able to identify new drugs against specific mutations and against specific tumor proteins and lipids. These drugs will hopefully be specific to preventing exosomes from preparing metastatic sites.

Our goal is to have limited side effects. As you know, we use a lot of harsh therapies such as chemotherapy that are based on cell replication and division, but so many cells in our bodies are already dividing, so we have plenty of side effects with the traditional therapy.

The aim of this study is not only to develop tests to predict who's going to have metastasis, but actually prevent and possibly treat patients with metastatic disease. There are very limited drugs for that right now for patients, once they have metastasis. For instance, when pancreatic cancer patients develop liver metastasis, less than 5% survive. For patients with brain metastasis, there's 0% survival. We hope the impact will be to prevent and to treat children with metastatic disease to various sites or potential sites of metastasis.

Although children have an overall lower incidence of cancer, compared with adults, once children get metastasis, it is quite aggressive, with a short latency period. You must therefore do something right away or the child will die very soon of metastatic disease.

That's in contrast to many adult cancers, which have a slower rate of metastasis such as prostate cancer that spreads to the bone or colon cancer that spreads to the liver. Children's cancers are very aggressive with a very high metastatic potential. I think the impact our work will be that we'll learn a lot more about how to prevent and treat metastatic disease in the pediatric population. That information could also help adults with metastatic potential and metastatic disease.

How will Weill Cornell Medicine collaborate with MSK in this study?

On our side, we're very good at analyzing the proteins and the lipids in our laboratory, but we need a laboratory to help us identify the mutational status. We could isolate the DNA and do some DNA testing, but our lab is not as sophisticated as Dr. Michael Berger's lab, where it is possible to identify probably the largest number of mutations out of the majority of labs in the country. By using his lab, we'll identify new mutations that have not previously been recognized in pediatric tumor exosomes and pediatric tumor, which will open the door for us to identify new drugs.

For instance, BRAF mutation is a mutation commonly seen in melanoma, but now we can measure the BRAF mutation in children with pediatric brain tumors. There might be some drugs already out there that we can identify and use in the pediatric population, but we’re also interested in identifying new mutations and developing novel drugs for the pediatric cancer patient.

What do you think the future holds for predicting how cancer spreads and what will this mean for patients?

I think it's an exciting time. In the past, so much emphasis was placed on the primary tumor and all the pharmaceutical companies based all their drugs on the primary tumor.

As we go forward and understand more about the biological steps, we're seeing that learning about the metastatic steps is very different to just learning about primary tumor biology. As our work and the work of others has shown, tumors are secreting a lot of tumor secreted factors such as growth factors and our work also has emphasized tumor secreted particles. We have to understand that cancer is a systemic disease; it's not just focused on the primary tumor.

I'm excited that people, drug companies and different granting agencies are now paying more attention to metastasis, which causes death in 90% of cases. More emphasis is being placed on the immune system, how it can go and how it can be re-educated where it actually helps promote cancer. There are bad immune cells that can be targeted. There are cells in the tumor microenvironment, so there are the secreted factors. There are a number of components that can be targeted now by understanding the metastatic cascade. I think there is a promising future.

Where can readers find more information?

About Dr. David Lyden

David C. Lyden is the Stavros Niarchos Professor of Pediatrics in the Pediatrics Department, Division of Hematology/Oncology at Weill Cornell Medicine and a pediatric Neuro-oncologist at the Memorial Sloan Kettering Cancer Center in New York City.

Dr. Lyden earned his PhD degree from the University of Vermont, his MD degree from Brown University, completing his Residency in Pediatrics at Duke University and fellowships and post-doctoral work at Memorial Sloan Kettering.

Early work in Dr. Lyden's laboratory resulted in several fundamental discoveries that involve the role of bone marrow-derived stem and progenitor cells in tumor vasculogenesis and in metastasis.

Dr. Lyden is a pioneer in the field of the metastasis and his discoveries have revolutionized our understanding of the metastatic cascade. Dr. Lyden demonstrated that secreted factors by the primary tumor prime certain tissues for tumor cell engraftment, defining the concept of the "pre-metastatic niche".

His laboratory has identified key proteins and the presence of nucleic acids in exosomes that support thrombosis generation, vascular leakiness, and pre-metastatic niche formation. Recently, he has defined the role of tumor exosomal integrins in organotropic metastasis.

Dr. Lyden is supportive and inspiring mentor to many young scientists, a sought-after speaker at metastasis and exosome biology meetings and the recipient of numerous honors and awards.

Among these, he was honored with the Career Achievement Award Belgium Society of Cell Biology and Development and the European Microenvironment Society Award, the Mina J. Bissel Award from the University of Porto, Portugal, the Presidential Award of Portugal, the Bial Medical Distinction Award and the Inaugural I.J. “Josh” Fidler Innovation in Metastasis Research Award from the Metastasis Research Society.

He is a member of the AACR Metastasis and Tumor Microenvironment Steering Committees, a Fellow of the Linnean Society of London and he holds a Visiting Professor Faculty Appointment at the Peking Union Medical School, Beijing, China. Dr. Lyden engages in full time research at WCM and treats children with brain malignancies at MSKCC.

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